Heart disease caused by the loss or dysfunction of cardiomyocytes is the leading cause of death worldwide. The adult mammalian heart possesses little regenerative potential and therefore displays fatal loss of function following myocardial infarction (MI) and other heart diseases. Fibrosis and scar formation due to activation of cardiac fibroblasts serve as barriers to cardiac regeneration and contribute to loss of contractile function, pathological remodeling and susceptibility to arrhythmias. Recently, combinations of cardiogenic transcription factors were shown to be capable of activating cardiac gene expression in fibroblasts in vitro. Moreover, we have shown that forced expression of four transcription factors in cardiac fibroblasts is sufficient to activate cardiac gene expression in vivo, leading to improvement of cardiac function and reduction of adverse ventricular remodeling following MI in mice. Although these reprogramming processes are not yet optimized, they hold great potential for eventual cardiac repair, avoiding many of the obstacles associated with cellular transplantation. The overall goals of this project are to further define the mechanisms involved in reprogramming of cardiac cell fate and to optimize the technology for reprogramming of fibroblasts to cardiomyocytes in vivo as a strategy for cardiac repair. We will also assess the ability of selected small molecules and microRNAs to enhance cardiac reprogramming in vivo as a strategy for restoration of cardiac function following MI. These studies will provide important new insights into the mechanisms of cardiac cell fate specification and differentiation and will pave the way toward innovative approaches for cardiac repair.